Summary of Work: Studies are designed toward the goal of providing insight into the cellular pathways leading to mutations in mammalian cells. Such an approach requires a well-defined assay system with a mutable target locus that can be readily isolated for molecular analyses. These studies are designed i) to further characterize already widely used in vitro mammalian mutagenesis assay systems, ii) to assess the role of specific mismatch repair pathways in the regulation of spontaneous and induced mutation, and iii) to develop new and more sensitive assays for detecting genomic alterations caused by environmental exposures in humans. Mutagenesis studies in mismatch repair deficient human cancer lines: Studies of mutation rates and molecular spectra at the endogenous hprt gene are continue using human cancer cell lines. These studies are designed to address the hypothesis that there exist functional differences among human mismatch repair (MMR) gene products that have specific effects on spontaneous and damage induced mutagenesis. Mutation rates at hprt were measured in human cancer cell lines defective in the MMR genes hMLH1, hPMS2, or GTBP, along with a cell line carrying mutations in both hMLH1 and hPMS2. These MMR-deficient cell lines exhibited a 50- to 750-fold increase in mutation rate relative to a MMR- proficient cancer cell line. From lowest to highest, the spontaneous mutation rates relative to the MMR-gene defects in the cell lines studied are as follows: hMLH1- < GTBP - < hPMS2 - < hMLH1 - /hPMS2 -. These data support the notion that MMR plays an important role in controlling the rate of spontaneous mutation and suggest that different MMR-gene defects vary in their ability to repair different types of DNA mismatches, thus leading to measurable quantitative differences in spontaneous mutagenesis. Additionally, a difference in mutation rates was observed between a hPMS2-defective cell line (3.1 x 10-5 mutations/cell/generation) and two hMLH1-defective cell lines (4.0 x 10-6 and 7.3 x 10-6 mutations/cell/generation). Defects in either the hPMS2 or hMLH1 genes should yield comparable mutation rates if the hPMS2 and hMLH1 gene products only function in the hMutLa heterodimer. These data suggest that hPMS2 plays a critical role in MMR, while additional hMLH1 homologues or hPMS2 alone may function to partially complement defects in hMLH1. Spontaneous mutant spectra are being generated in cell lines carrying mutations in each of the MMR genes described above in order to characterize the molecular effects of specific MMR defects. These data support a redundancy of function for the hMutSa and hMutSb repair complexes. We are now evaluating the ability DNA damage to illicit a hyper-mutagenic response of MMR-deficient. The status of p53 in L5178Y Tk+\- - 3.7.2C mouse lymphoma cells: These studies involve the further characterization of a cell line widely used as an in vitro mammalian mutagenesis gene-tox testing systems. In the past year, we have defined mutations in both alleles of the Trp53 gene in the L5178Y Tk +\- - 3.7.2C mouse lymphoma cells. We first defined the PCR conditions for optimal amplification of p53 exons 3-9. Western blot and SSCP analysis were performed and data suggest that these cells carry two different p53 mutations. We have used independently derived subclones of L5178Y cells to provide a molecular definition of p53 gene status in this cell line. In addition, we have used L5178Y TK-/- mutant subclones, which have lost one mouse chromosome 11 (the site of both the tk and p53 genes) in order to define allele and chromosome specific p53 mutations in the L5178Y cell line.